The present invention relates to haptens that are used for the preparation of immunogens, antibodies and conjugates for use in competitive immunoassays for the detection of the major LSD metabolite, 2-oxo-3-hydroxy-LSD.
The present invention also relates to a method and kit for detecting or determining 2-oxo-3-hydroxy-LSD. The method and kit of the present invention are intended not to significantly cross-react with parent LSD itself or with nor LSD.
By “detecting” is meant qualitatively analysing for the presence or absence of a is substance.
By “determining” is meant quantitatively analysing for the amount of a substance.
Lysergic acid diethylamide (LSD) (FIG. 1, 1) is a powerful, psychoactive compound. It is classified as a Schedule I drug. The compound is available in pills, solutions and impregnated sugar cubes, blotting paper or vitamin tablets.
LSD is a very potent hallucinogen, 10–150 times as potent as psilocybin and 4500–9275 times as potent as mescaline. The isomeric compound d-iso-LSD (FIG. 1, 2) is inactive.
With increasing restrictions on administration of LSD to human subjects, current knowledge on the distribution, metabolic profile and extraction of LSD in man is limited. A considerable number of reports are available on the distribution and metabolic profile of LSD in animals where the most common metabolites found are nor-LSD (FIG. 1, 3), 2-oxo-3-hydroxy-LSD (FIG. 1, 4), 2-oxo-LSD, 13-hydroxy-LSD, 14-hydroxy-LSD, N-desethyl-LSD, N-ethyl-N-(2-hydroxyethyl)-LSD (amide N), N-ethyl-N-vinyl-LSD (amide N) and lysergic acid. The major metabolites are nor-LSD 3 and 2-oxo-3-hydroxy LSD 4, the latter of which was only recently detected in human urine submitted for drug testing, its identity having been confirmed by comparing LC-MS characteristics with a reference compound. The average concentration of 2-oxo-3-hydroxy-LSD 4 was 20 times more than that of LSD. In routine analysis of human urine, d-iso-LSD 2 was also detected. This compound is believed to be a by-product from the illicit preparation of LSD.
Due to the very low dose consumed (usually 40 to 120 μg) and due to rapid metabolism with less than 1% excreted unchanged in urine, identification of LSD in biological samples is a major challenge to forensic scientists. Furthermore, the instability of LSD in acid, heat and light has made its identification even more challenging. Because LSD is metabolized to a number of compounds, most known methods are aimed at identifying unchanged (or parent) LSD in biological samples.
Although LSD is most commonly detected in urine by GC-MS, immunoassays, particularly competitive binding immunoassays, would be the simplest and most time-saving screening methods available. Competitive binding immunoassays, as their name implies, measure competition in binding to antibody between a fixed amount of labeled antigen, the ‘detection reagent’ (or conjugate), and an unknown quantity of unlabelled antigen, the ‘sample’.
Commercial immunoassay methods for LSD include radioimmunoassay procedures which are very sensitive, but do require radionuclide tracers, for example 125I and 3H, and, in some cases, a preliminary extraction step. For urine drug testing by radioimmunoassay, samples are identified as positive or negative by comparing the counts with that of a cut-off standard containing 500 pg/ml of LSD.
Nonisotopic homogeneous immunoassays for LSD are also commercially available. The Cloned Enzyme Donor Immunoassay (CEDIA, Boehringer Mannheim) and Enzyme Multiplied Inmunoassay (EMIT, Behring Diagnostics) are based on the principle of enzyme activation. The Online Immunoassay (Roche Diagnostic Systems) is based on kinetic interaction of microparticles in solution. These three assays are specially designed for large-scale analysis or automated analyzers. Microplate Immunoassay (STC Diagnostics) is available for small-scale testing. These nonisotopic LSD immunoassays correlate well with the original LSD radioimmunoassays.
All currently commercially available LSD immunoassay methods are specific for the parent drug, LSD, and generally exhibit low cross-reactivity with LSD metabolites.
For example, U.S. Pat. No. 6,207,396 B1 (Microgenics Corporation) discloses haptens that are derivatives of the LSD parent drug (not 2-oxo-3-hydroxy LSD), through the indole ring N-1 position. The antibodies of U.S. Pat. No. 6,207,396 B1 are specific to d-LSD and do not cross-react well with 2-oxo-3-hydroxy-LSD (1.82%) or with iso-LSD (0.04%) (see Table 1).
Examination of the prior art fails to reveal an antibody specific for 2-oxo-3-hydroxy LSD. EP 1 148 339 A2 (Roche Diagnostics Corporation) discloses haptens derivatised at position N-1, either of the indole ring of 2-oxo-3-hydroxy-LSD or of 2-oxo-LSD. The antibodies obtained in EP 1 148 339 A2 displayed high levels of cross-reactivity, when compared to 2-oxo-3-hydroxy-LSD, to the parent drug LSD (74.9–84.4%) and low cross-reactivity to the second metabolite, nor-LSD (1.8–4.6%) (paragraph 71).
EP 0 816 364 A1 (F. Hoffmann-La-Roche AG) and Bioconjugate Chemistry 1997, 8, pp 896–905 each describe the preparation of haptens either at the N-1 position of the indole ring of LSD itself or from nor LSD. Bioconjugate Chemistry 1997, 8, 896–905 discloses that antibodies generated to immunogen 4 displayed 100% cross-reactivity with d-LSD, 20% cross-reactivity with nor-LSD and 50% cross-reactivity with 2-oxo-3-hydroxy-LSD. Antibodies generated to immunogen 8 displayed 100% cross-reactivity with LSD, 40% cross-reactivity with nor-LSD and <20% cross-reactivity with 2-oxo-3-hydroxy-LSD.
Clin. Chem. 23/2, 169–174 (1977) describes a radioimmunoassay for LSD in serum and urine, employing antisera to two different immunogens, in which LSD itself is derivatised either at the indole nitrogen atom or via the nitrogen of the 8β-carboxamide.
The present inventors are unaware of any haptens derivatised with a crosslinker at the nitrogen of the 8β-carboxamide of 2-oxo-3-hydroxy-LSD.
The present inventors are also unaware of antibodies specific for 2-oxo-3-hydroxy-LSD but lacking significant cross-reactivity to parent LSD itself or to nor LSD.